1. Field of the Invention
The invention relates to compositions and methods of using nucleic acids and amino acids that encode enzymes involved in the synthesis of glucosinolates (GSL). The invention is exemplified by nucleic acids and amino acids encoding a BoGSL-ALK, an enzyme involved in glucosinolate side-chain desaturation, isolated from Brassica oleracea (Groups Botrytis (Cauliflower and Broccoli), Italica (Broccoli) and Viridis (Collard)), and methods of using these sequences to increase or decrease the levels of alkenyl glucosinolates in Brassica oleracea plant varieties. The invention also relates to methods and compositions for marker assisted gene selection, exemplified by primers and methods of using them for selecting BoGSL-ALK and two other key genes in GSL synthesis, BoGSL-PRO and BoGSL-ELONG.
2. Introduction
GSL are a diverse class of thioglucosides that are synthesized by many species of the order Capparales, including Brassica and Arabidopsis Heynh. The GSL molecule consists of two parts; a common glycone moiety and a variable aglycone side chain (Fenwick et al., Crit. Rev. Food Sci. Nutr., 18:123-301 (1983); Rosa et al., Hort Rev, 19:99-215 (1997)). The aglycone part may contain aliphatic, indolyl, or aromatic side chains and is derived from a corresponding α-amino acid. In the general GSL biosynthetic pathway proposed by Underhill, Glucosinolates. Encyclopedia of Plant Physiology (New Series), Vol. 8, Springer Verlag, Berlin, pp. 493-511 (1980); Larsen, Glucosinolates. In: The Biochemistry of Plants (EE Conn, ed.), Vol. 7, Academic Press, New York, pp. 501-525 (1981); and Haughn et al., Plant Physiol, 97:217-226 (1991), aliphatic GSL are derived from methionine. Genetic studies in Arabidopsis thaliana (Mithen et al., Heredity, 74:210-215 (1995); Mithen and Campos, Entomol. Exp. Appl., 80:202-205 (1996)) and Brassica sp. (Magrath et al., Plant Breeding, 111:55-72 (1993); Magrath et al., Heredity, 72:290-299 (1994)) support the biochemical pathway proposed for biosynthesis of aliphatic GSL. The synthesis of these compounds is determined by a simple genetic system containing two distinct sets of genes, one determining side-chain elongation and the second one for chemical modification of the side-chains. Aliphatic GSL profiles vary considerably in A. thaliana ecotypes and in Brassica crops and species. These GSL are synthesized in the following sequence: methylsulfinylalkyl, alkenyl and hydroxy-types, which can be divided into three-carbon (3C) four-carbon (4C) and five-carbon (5C) groups based on their side-chain length.
A number of studies suggest that consumption of vegetables, in particular, crops such as broccoli [Brassica oleraca (Italica Group)] and other crucifers, reduces the incidence of cancer in humans and other mammals (Block et al., Nutr. Cancer, 18:1-29 (1992); Fahey and Talalay (1998) pp. 16-22. In: T. Shibamoto, J. Terao and T. Osawa (eds.). Functional foods for disease prevention I. Fruits, vegetables and teas., Amer. Chem. Soc. Symp. Ser., 701. Amer. Chem. Soc., Washington, D.C.; Prochaska et al., Proc. Natl. Acad. Sci. USA, 89:2394-2398 (1992)). This seems to be due to the presence of inducers of phase II enzymes, that detoxify carcinogens and mutagens in various mammalian organs (Prestera et al., Adv. Enzyme Regulat., 33:28 1-296 (1996); Prochaska et al., (1992) supra; Talalay et al., pp. 469-478 (1992). In: L. W. Wattenberg, M. Lipkin, C. W. Boone, and G. J. Kelloff, (eds.). Cancer chemoprevention, CRC Press, Boca Raton, Fla.). In broccoli, the isothiocyanate sulfurophane, derived from the GSL glucoraphanin by the action of the enzyme myrosinase, was identified as a potent inducer of these enzymes, conferring protection against mammary tumor growth in rats after treatment with dimethyl benzanthracene, a carcinogenic agent (Zhang et al., Proc. Natl. Acad. Sci. USA, 91:3147-3150 (1994)). Glucoraphanin is one of the major GSL present in some crops of B. oleracea such as broccoli (Farnham et al., J. Amer. Soc. Hort. Sci., 125:482-488 (2000)) cauliflower, [B. oleraca (Botrytis Group)], cabbage [B. oleraca (Capitata Group)] and brussels sprouts [B. oleraca (Gemmifera Group)] (Rosa et al., Hort Rev, 19:99-215 (1997)). Unfortunately, the wide range of glucoraphanin in broccoli can lead to consumer confusion. At the present time the consumer that eats broccoli might assume that it has high sulfurophane content and that therefore this produce is conferring health benefits by carcinogen detoxification. However, this is not always true. For example, the heads of broccoli inbred plants have been found to contain from 0.28 to 4.0 mol/gram of fresh weight glucoraphanin content (Farnham, et al. (2000) J. Amer. Soc. Hort. Sci. 125(4):482-488). Because most of the existing broccoli varieties in the supermarket have not been selected for glucoraphanin content, the precursor of sulfurophane, certain produce could actually possess only small concentrations of this compound and in reality be ineffective for its assumed anticarcinogenic properties.
Although certain GSL derivatives have a protective effect against cancer (Rosa et al., (1997) supra), there are some that may have detrimental effects such those derived from alkenyl GSL in rapeseed seed meal (Brassica napus L.). Such GSLs act as anti-nutrients affecting not only animal growth and development, but also lowering food intake. Additionally, modified isothiocyanates from the aliphatic GSL progoitrin may have goitrogenic effects in animals (Rosa et al., (1997) supra). There is therefore an interest in methods and compositions for lowering the amount of these antinutritional GSL and for providing crucifer varieties with consistently increased amounts of protective GSL in plants intended for human and animal consumption.
The polymerase chain reaction (PCR) is widely used in genomic DNA analysis. One of its main applications has been on the development of DNA markers for map construction, which are useful in breeding, taxonomy, evolution and gene cloning. Several PCR marker systems are available varying in complexity, reliability and information generating capacity. These include random amplified polymorphic DNA (RAPD), simple sequence repeat polymorphism (SSR), amplified fragment length polymorphism (AFLP) and a few others (Lee, Adv. Agronomy, 55:265-344 (1995); Rafalski et al., in Non Mammalian genome analysis: A practical guide, Acad. Press, pp. 75-134 (1996)). Each system has its own advantages and disadvantages. For example, RAPD is a simple method to fingerprint genomic DNA, but poor consistency and low multiplexing output limit its use. SSR has the advantage that it produces mostly co-dominant markers, however the development of these is expensive and time-consuming. AFLPs are now widely used for a variety of applications due to their high multiplexing ratio (Vos et al., Nucleic Acids Res, 23:4407-4414 (1995)). The main disadvantage of this method is its complexity, it being necessary to perform multiple steps including DNA digestion, ligation and amplification, which makes it difficult to optimize the conditions for each step. Furthermore, methylation of genomic DNA can result in pseudo polymorphism when the restriction enzyme used is methylation-sensitive. Also the use of the MseI restriction enzyme, which recognizes AATT restriction sites, often results in uneven marker distribution in the genome of some species (Haanstra et al., Theor Appl Genet, 99:254-271 (1999)). Ability to isolate specific bands for sequencing is another concern when selecting a marker system, especially for the development of new markers for gene tagging. In most cases, both RAPD and AFLP markers need to be cloned into vectors, which adds to the labor. In addition, for AFLP bands it is notoriously difficult to isolate the correct fragment due to band overlapping. Therefore, there is a need for a PCR marker system that combines the desired attributes of simplicity, reliability, moderate throughput ratio, facile sequencing of selected band, with targeting of coding sequences in the genome and efficient identification of a moderate number of co-dominant markers.
3. Relevant Literature
Brassica napus seeds, and plants producing them, obtained by genetic mutation and having a maximum total glucosinolate content of about 3.4 micromoles per gram of seed and a maximum 4-hydroxy-3-indolylmethyl glucosinolate content of 1.9 micromoles per gram of seed are disclosed in U.S. Pat. No. 6,225,533.
In Arabidopsis thaliana, several genes involved in the glucosinolate pathway have been identified by genetic analysis (Mithen et al., Heredity, 74:210-215 (1995); Mithen and Campos, Entomol. Exp Appl., 80:202-205 (1996)). In rapeseed, genes regulating aglycon side-chain elongation and modification have been reported (Magrath et al., Plant Breeding, 111:55-72 (1993); Parkin, et al. (1994) Heredity 72:594-598 (1994)). Campos de Quiros et al., Theoretical and Applied Genetics, 101:429-437 (2000)) disclose the mapping and sequencing of a GSL-ELONG gene from A. thaliana. However, many steps in side-chain elongation, glycone formation, and aglycone modification remain to be characterized biochemically and genetically.
Hall et al., Theoretical and Applied Genetics, 102:369-374 (2001)) disclose the fine mapping of the OHP locus in A. thaliana, which contains a gene cluster of three open reading frames with high homology to sequences encoding 2-oxoglutarate-dependent dioxygenases (2-ODDs). The 2-ODD translation products associate with glucosinolate hydroxylation activity, and Hall speculates that the ALK and OHP loci in A. thaliana may either represent two closely linked genes or different alleles of the same gene. Hall et al. do not provide either a nucleic acid or an amino acid sequence. Kliebenstein et al., Plant Cell, 13:681-693 (2001)) disclose the identification of genes encoding two 2-oxoglutarate-dependent dioxygenases from Arabidopsis that control the conversion of methylsulfinylalkyl glucosinolate to either the alkenyl or the hydroxyalkyl form. Kliebenstein et al. expressed these genes in E. coli to determine their catalytic activity. WO 99/27120 discloses cloning of GSL-ELONGASE gene alleles from Arabidopsis. 